CN109880827A - The method for building up of hepatolenticular degeneration zebra fish model - Google Patents
The method for building up of hepatolenticular degeneration zebra fish model Download PDFInfo
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Abstract
The invention discloses the method for building up of hepatolenticular degeneration zebra fish model, including the following steps: by the design of gRNA target sequence on the amino acid domain that zebra fish ATP7B gene extron 2 encodes, synthetic primer；Using pMD19-gata5_gRNA scaffold carrier as template, using primer preparation gRNA in-vitro transcription template is transcribed in vitro, purification and recovery obtains gRNA；GRNA and Cas9 mRNA is injected into one cell stage zebrafish embryo, culture screening is to obtain ATP7B mutation zebra fish, and the present invention is for simulating Wilson disease feature.
Description
Technical field
The present invention relates to the construction method technical field of model animal disease model more particularly to hepatolenticular degeneration zebras
The method for building up of fish model.
Background technique
Hepatolenticular degeneration (also known as Wilson disease, OMIM#277900) is since copper ion is excessively accumulated in liver
Cell causes hepatocellular injury, and excessive copper ion enters brain by blood-brain barrier after liver cell is flowed out, and is deposited on beans shape
Core position then causes the site tissue damage.The global incidence of the disease is about 1/30,000, and the disease incidence of asian population is high
In American-European crowd, East Asia Region is up to 1/10,000.The clinical manifestation of hepatolenticular degeneration is with hepatopathy and neuropsychic symptom
Main, there is endocrine and disease in the blood system in small number of patients.Liver anomalies first appear as fatty change, can then progress to acute
Liver failure, hepatitis and liver fibrosis.Basal ganglion and brain stem are that copper accumulation damages another easy hair position, liver beans shape simultaneously
Nuclear degeneration patient nervous symptoms the most apparent are Parkinson's sample movement defect and epilepsy.Therefore how liver lenticular nucleus is effectively treated
Denaturation has very important realistic meaning.
Copper ion metabolic imbalance is the feature performance of Wilson disease.Under normal circumstances, human body is obtained from diet
Copper ion is obtained, liver is entered by portal circulation after small intestinal absorption.Liver is the center place for maintaining copper ion stable state.
ATP7B is a kind of metal transport p-type atriphos (ATP) enzyme, research shows that ATP7B gene mutation causes the protein function to drop
Low or missing, copper ion cannot be transported to around liver plasma membrane through golgiosome, to prevent copper ion and blood plasma covellite egg
White combination and the excretion through bile cause copper to be accumulated in liver cell.Liver is body copper excessively most important afflicted organ,
Studies have shown that 100% patient has hepatopathy symptom in the clinical manifestation of hepatolenticular degeneration patient, 40-50% patient has nerve
Symptom, 20% patient have the exception of other organs.Although excessively how multiplicity to be induced to face about copper at present by years of researches
Bed performance is not also completely clear.
Dong etc. carries out ATP7B gene Sanger sequencing analysis, discovery to 632 hepatolenticular degeneration patients of Chinese Han nationality
90% (569/632) patient has two or more harmful variation.Up to the present, reported liver lenticular nucleus becomes for cut-off
Property patient's ATP7B genetic mutation type is up to more than 800 kinds.However, the disease phenotype of hepatolenticular degeneration and ATP7B genotype are but not
It is simple corresponding relationship.Ferenci etc. has found facing for ATP7B genotype and patient to 1357 patient clinical phenotypic analyses
Bed phenotype is uncorrelated.It is found by multinomial logistic regression, PNPLA3G allele (rs738409) rather than ATP7B gene
Type or liver copper content, be in patient/severe liver fat becomes the independent hazard factor of (> 33%ofhepatocytes).Liver beans
The relevant liver cirrhosis patient of shape nuclear degeneration has higher splenomegaly risk (odds relative to the relevant liver cirrhosis patient of HBV
Ratio=4.15,95%confidence interval:1.38-12.45, P=0.011).
The disease process of hepatolenticular degeneration enters nucleus by cytoplasm along with a large amount of copper ions, thus opens thin
The remodeling of born of the same parents' transcript profile.After the discoveries such as Hamilton activate LXR/RXR signal path, when the accumulation of liver copper does not become, still may be used
Improve ATP7B-/-Mouse liver lipid-metabolism symptom.It is found by experiment in vitro, inhibits p38 and c-Jun signal path, it can be extensive
It is multiple that liver cell copper homeostasis caused by (i.e. H1069Q) is mutated as mild type ATP7B.ATP7B is not only expressed in liver cell,
Also expression becomes studies have shown that liver cell specific knockdown ATP7B only results in liver fat without inflammation hair in nonparenchymal cell
It is raw.Thus, it is believed that in hepatolenticular degeneration generating process, the accumulation of copper caused by ATP7B is mutated is cause, excessive copper
Ion enter after nucleus open transcript profile remodeling be hepatolenticular degeneration multiplicity clinical phenotypes immediate cause.
At present researcher about copper homeostasis cause hepatolenticular degeneration occur downstream molecules mechanism explore still in
Starting stage, and there is an urgent need to explore copper by the animal model of multiplicity for the existing uncertainty for driving copper therapeutic agent and hidden danger
The molecular regulation mechanism that homeostasis causes hepatolenticular degeneration to occur.This research and utilization CRISPR/Cas9 system constructs ATP7B
Zebra fish system, by the morbidity feelings for accumulating the analyses such as situation, copper sensibility, hepatic pathology description mutation fish system to mutant copper
Condition.
Summary of the invention
The method for building up of present invention offer hepatolenticular degeneration zebra fish model.
The scheme of the invention is:
The method for building up of hepatolenticular degeneration zebra fish model, including the following steps:
S1, gRNA target sequence is designed on the amino acid domain that zebra fish ATP7B gene extron 2 encodes, is closed
At primer；
S2, using pMD19-gata5_gRNA scaffold carrier as template, use primer in step S1 to prepare gRNA body
Outer transcription templates；
S3, the gRNA in-vitro transcription template of step S2 is transcribed in vitro, purification and recovery acquisition gRNA；
S4, microinjection after step S3 recycling acquisition gRNA and Cas9mRNA fresh mix is entered into one cell stage zebra fish embryo
Tire, each target spot inject 180~220 pieces；
S5, collection step S4 are injected into the zebrafish embryo after one cell stage zebrafish embryo is completed 48 hours, extract base
Because of group, PCR amplification target fragment, sequencing, sequencing result is that bimodal injection batch embryo enters feeding system, raising to property at
Effective mutation type zebra fish is detected by screening its next-generation embryo when ripe, to obtain ATP7B mutation zebra fish；
S6, the ATP7B of step S5 is taken to be mutated zebra fish progress ATP7B expression quantity detection；
S7, the ATP7B of step S5 is taken to be mutated zebra fish, respectively to the ATP7B mutation spot for starting to feed 5 days and feed 45 days
Horse fish carries out copper content analysis；
S8, the ATP7B of step S5 is taken to be mutated zebra fish progress adult fish Shape analysis, the adult fish is 8 months big ATP7B
It is mutated zebra fish；
S9, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out copper and store analysis, and the copper stores analysis and includes
The micro- sem observation situation of light field and ICP-MS quantify copper content；
S10, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out the analysis of copper sensibility concentration dependant and disease
Reason analysis；
S11, analysis description is carried out by obtaining data to step S6, step S7, step S8, step S9, step S10
The incidence of ATP7B mutation zebra fish.
GRNA target sequence is designed in zebra fish ATP7B gene extron in the step S1 as a preferred technical solution,
The amino acid domain of 2 coding of son.
As a preferred technical solution, in the step S4 after gRNA and Cas9mRNA fresh mix microinjection enter it is slender
Born of the same parents' phase zebrafish embryo, gRNA are 300~600pg, Cas9mRNA 200pg.
Each target spot injects 200 pieces in the S4 step as a preferred technical solution,.
The micro- sem observation situation method of light field in the step S9 as a preferred technical solution: ATP7B in step S5 is taken
The juvenile fish that mutation zebra fish has just hatched is dipped into copper solution, light field microscopically observation to zebra fish mutant liver and head
Portion's nigrescence situation.
The method that ICP-MS quantifies copper content in the step S9 as a preferred technical solution: 1000 are taken to take step
5 -day-old of ATP7B is mutated zebra fish juvenile fish in S5 or 5 take 45 -day-old in step S5 of ATP7B to be mutated zebra fish youth fish, adds
Enter HClO4Digested 10 hours in 180 DEG C, after be transferred to 120 DEG C of shake residual acid made to volatilize, diluted with Milli-Q high purity water, use
ICP-MS measures each tissue copper content.
The analysis of copper sensibility concentration dependant is uses in the step S10 as a preferred technical solution, light field microscope
Under observe the phenotype of body rollover, distortion that zebra fish mutant shows in copper-containing solution and for statistical analysis.
Hepatic pathology analysis is young using ATP7B is mutated zebra fish in the step S10 as a preferred technical solution,
Fish soaking samples after 24 hours in copper-containing solution, fixed, dehydration, transparent, waxdip, embedding.Slice thickness is 4 μm when slice,
HE dyeing, the micro- sem observation hepatic pathology situation of light field.
The foundation side of hepatolenticular degeneration zebra fish model described in step S9-S10 is required as a preferred technical solution,
Method, the ATP7B mutation zebra fish juvenile fish are that development is extremely big to 5-7.
Due to the method for building up using above-mentioned hepatolenticular degeneration zebra fish model, including the following steps:
S1, gRNA target sequence is designed on the amino acid domain that zebra fish ATP7B gene extron 2 encodes, is closed
At primer；
S2, using pMD19-gata5_gRNA scaffold carrier as template, use primer in step S1 to prepare gRNA body
Outer transcription templates；
S3, the gRNA in-vitro transcription template of step S2 is transcribed in vitro, purification and recovery acquisition gRNA；
S4, microinjection after step S3 recycling acquisition gRNA and Cas9mRNA fresh mix is entered into one cell stage zebra fish embryo
Tire, each target spot inject 180~220 pieces；
S5, collection step S4 are injected into the zebrafish embryo after one cell stage zebrafish embryo is completed 48 hours, extract base
Because of group, PCR amplification target fragment, sequencing, sequencing result is that bimodal injection batch embryo enters feeding system, raising to property at
Effective mutation type zebra fish is detected by screening its next-generation embryo when ripe, to obtain ATP7B mutation zebra fish；
S6, the ATP7B of step S5 is taken to be mutated zebra fish progress ATP7B expression quantity detection；
S7, the ATP7B of step S5 is taken to be mutated zebra fish, respectively to the ATP7B mutation spot for starting to feed 5 days and feed 45 days
Horse fish carries out copper content analysis；
S8, the ATP7B of step S5 is taken to be mutated zebra fish progress adult fish Shape analysis, the adult fish is 8 months big ATP7B
It is mutated zebra fish；
S9, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out copper and store analysis, and the copper stores analysis and includes
The micro- sem observation situation of light field and ICP-MS quantify copper content；
S10, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out the analysis of copper sensibility concentration dependant and disease
Reason analysis；
S11, analysis description is carried out by obtaining data to step S6, step S7, step S8, step S9, step S10
The incidence of ATP7B mutation zebra fish.
Advantages of the present invention:
(1) using ATP7B mutation zebra fish as experimental animal, the disease model and human diseases similitude of duplication are good,
The mechanism with similar human diseases feature can more accurately be induced；
(2) at low cost, the period is short, reproducible, highly reliable, can be effectively reduced pharmacological experiment and drug screening at
This, further investigation hepatolenticular degeneration manages mechanism, finds new treatment means for hepatolenticular degeneration；
(3) facilitate the regularity of occurrence and development of understanding hepatolenticular degeneration, to push hepatolenticular degeneration medical industry
Development provides strong basis.
Detailed description of the invention
Fig. 1 is zebra fish ATP7B mutation construction；
Fig. 2 is zebra fish ATP7B mutant copper content and body shape changes；
Fig. 3 is that zebra fish ATP7B mutant increases copper sensibility.
Specific embodiment
In order to make up the above deficiency, on the present invention provides the method for building up of hepatolenticular degeneration zebra fish model to solve
State the problems in background technique.
The method for building up of hepatolenticular degeneration zebra fish model, including the following steps:
S1, gRNA target sequence is designed on the amino acid domain that zebra fish ATP7B gene extron 2 encodes, is closed
At primer；
S2, using pMD19-gata5_gRNA scaffold carrier as template, use primer in step S1 to prepare gRNA body
Outer transcription templates；
S3, the gRNA in-vitro transcription template of step S2 is transcribed in vitro, purification and recovery acquisition gRNA；
S4, microinjection after step S3 recycling acquisition gRNA and Cas9mRNA fresh mix is entered into one cell stage zebra fish embryo
Tire, each target spot inject 180~220 pieces；
S5, collection step S4 are injected into the zebrafish embryo after one cell stage zebrafish embryo is completed 48 hours, extract base
Because of group, PCR amplification target fragment, sequencing, sequencing result is that bimodal injection batch embryo enters feeding system, raising to property at
Effective mutation type zebra fish is detected by screening its next-generation embryo when ripe, to obtain ATP7B mutation zebra fish；
S6, the ATP7B of step S5 is taken to be mutated zebra fish progress ATP7B expression quantity detection；
S7, the ATP7B of step S5 is taken to be mutated zebra fish, respectively to the ATP7B mutation spot for starting to feed 5 days and feed 45 days
Horse fish carries out copper content analysis；
S8, the ATP7B of step S5 is taken to be mutated zebra fish progress adult fish Shape analysis, the adult fish is 8 months big ATP7B
It is mutated zebra fish；
S9, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out copper and store analysis, and the copper stores analysis and includes
The micro- sem observation situation of light field and ICP-MS quantify copper content；
S10, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out the analysis of copper sensibility concentration dependant and disease
Reason analysis；
S11, analysis description is carried out by obtaining data to step S6, step S7, step S8, step S9, step S10
The incidence of ATP7B mutation zebra fish.
GRNA target sequence designs the amino acid structure encoded in zebra fish ATP7B gene extron 2 in the step S1
Domain.
Microinjection enters one cell stage zebrafish embryo, gRNA after gRNA and Cas9mRNA fresh mix in the step S4
For 300~600pg, Cas9mRNA 200pg.
Each target spot injects 200 pieces in the S4 step.
The micro- sem observation situation method of light field in the step S9: ATP7B mutation zebra fish in step S5 is taken just to hatch
Juvenile fish be dipped into copper solution, light field microscopically observation to zebra fish mutant liver and head nigrescence situation.
The method that ICP-MS quantifies copper content in the step S9: take 1000 5 -day-old in step S5 of ATP7B is taken to be mutated
Zebra fish juvenile fish or 5 take 45 -day-old in step S5 of ATP7B to be mutated zebra fish youth fish, and HClO is added410 are digested in 180 DEG C
Hour, after be transferred to 120 DEG C of shake residual acid made to volatilize, diluted with Milli-Q high purity water, contained with each tissue copper of ICP-MS measurement
Amount.
To use, light field microscopically observation to zebra fish is mutated the analysis of copper sensibility concentration dependant in the step S10
The phenotype of body rollover, distortion that body is shown in copper-containing solution is simultaneously for statistical analysis.
ATP7B mutation zebra fish juvenile fish is immersed in copper-containing solution by hepatic pathology analysis in the step S10 to use,
It is sampled after 24 hours, fixed, dehydration, transparent, waxdip, embedding.Slice thickness is 4 μm when slice, and HE dyeing, light field microscope is seen
Examine hepatic pathology situation.
It is required that the method for building up of hepatolenticular degeneration zebra fish model described in step S9-S10, the ATP7B mutation spot
Horse fish juvenile fish is that development is extremely big to 5-7.
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is further explained.
Embodiment 1:
1. there are the amino acid region that zebra fish ATP7B gene extron 2 encodes 3, which to be spaced the copper to connect, combines conservative sequence
Arrange GMX1CX2X3CV, first MBD (metalbinding in Bioinformatics Prediction exons coding protein structure domain
domain).And mankind's ATP7B albumen MBD structural domain is the high-incidence region of mutation, therefore the design of CRISPR/Cas9 action target spot is existed
Exon 2 region can simulate the onset state of mankind's disease as far as possible.By the micro- note of gRNA and Cas9mRNA fresh mix
It penetrates, has collected 48 hours embryos after injection, be sequenced after PCR amplification target fragment, there is bimodal appearance in F0 generation as the result is shown, illustrates this
The CRISPR/Cas9 system of secondary design works (Fig. 1, A- control group, B- injection group), cultivates and screens by F1 generation, final
It is (Fig. 1 C) to four kinds of efficient gene editor fishes, it is (Fig. 1 C, black that two of them, which has obtained F2 for homozygous genotype zebra fish,
It irises out).
Embodiment 2:
Mutant copper content and shape: in homozygous genotype zebra fish system, QPCR is tied the F2 screened in embodiment 1
Fruit shows that atp7b expression quantity significantly reduces (Fig. 2A), and mutant copper content is aobvious when developed as the result is shown by ICP-MS by 45 days
It writes and is higher than wild type fish (Fig. 2 B).Develop the adult mutant big by 8 months show small and short shape (Fig. 2 C, it is +/-: miscellaneous
Close genotype；/-: homozygous genotype).
Embodiment 3
Mutant copper sensibility: the F2 screened in Example 1 is shown for homozygous genotype zebra fish juvenile fish, ICP-MS
Copper is lower than wild type fish in mutant at this time, but does not have conspicuousness (Fig. 3 A), because zebra fish juvenile fish is at this time mainly from yolk
Rather than external world's food acquisition nutrition is related.It is dipped into copper solution when juvenile fish, light field microscope observes mutant liver
The copper of accumulation is higher than wild type (Fig. 3 B, arrow), and ICP-MS shows that the copper content of mutant accumulation in copper ring border is higher than wild type
(Fig. 3 C).Light field microscopically observation shows the phenotype (figure of more body rollovers, distortion to mutant in copper-containing solution
3D, arrow).Statistical data is shown, between 1 μM to 5 μM, the ratio of mutant performance travelling obstacle is significantly higher than wild type fish
(Fig. 3 E).HE coloration result shows that mutant liver shows more vesicle fat changes and nucleus in copper ring border
Swelling (Fig. 3 F).
Integrated embodiment 2 and embodiment 3, thus ATP7B mutation zebra fish system established by the present invention can simulate liver beans
Shape nuclear degeneration genius morbi.
The above shows and describes the basic principle, main features and advantages of the invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent circle.
Claims (9)
1. the method for building up of hepatolenticular degeneration zebra fish model, characterized in that it comprises the following steps:
S1, gRNA target sequence is designed on the amino acid domain that zebra fish ATP7B gene extron 2 encodes, synthesis is drawn
Object；
S2, using pMD19-gata5_gRNA scaffold carrier as template, using in step S1 primer preparation gRNA turn in vitro
Record template；
S3, the gRNA in-vitro transcription template of step S2 is transcribed in vitro, purification and recovery acquisition gRNA；
S4, microinjection after step S3 recycling acquisition gRNA and Cas9 mRNA fresh mix is entered into one cell stage zebrafish embryo,
Each target spot injects 180~220 pieces；
S5, collection step S4 are injected into the zebrafish embryo after one cell stage zebrafish embryo is completed 48 hours, extract genome,
PCR amplification target fragment, sequencing, sequencing result are that bimodal injection batch embryo enters feeding system, are raised when sexal maturity
Effective mutation type zebra fish is detected by screening its next-generation embryo, to obtain ATP7B mutation zebra fish；
S6, the ATP7B of step S5 is taken to be mutated zebra fish progress ATP7B expression quantity detection；
S7, it takes the ATP7B of step S5 to be mutated zebra fish, zebra fish is mutated to the ATP7B for starting feed 5 days and feeding 45 days respectively
Carry out copper content analysis；
S8, the ATP7B of step S5 is taken to be mutated zebra fish progress adult fish Shape analysis；
S9, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out copper and store analysis, and it includes light field that the copper, which stores analysis,
Micro- sem observation situation and ICP-MS quantify copper content；
S10, the juvenile fish for taking the ATP7B of step S5 to be mutated 6 -day-old of zebra fish carry out the analysis of copper sensibility concentration dependant and pathology point
Analysis；
S11, it is dashed forward by carrying out analysis description ATP7B to step S6, step S7, step S8, step S9, step S10 acquisition data
Become the incidence of zebra fish.
2. the method for building up of hepatolenticular degeneration zebra fish model as described in claim 1, it is characterised in that: the step S1
It is middle that gRNA target sequence is designed to the amino acid domain encoded in zebra fish ATP7B gene extron 2.
3. the method for building up of hepatolenticular degeneration zebra fish model as described in claim 1, it is characterised in that: in the S4
Microinjection enters one cell stage zebrafish embryo after gRNA and Cas9 mRNA fresh mix, and gRNA is 300~600pg, Cas9
MRNA is 200pg.
4. the method for building up of hepatolenticular degeneration zebra fish model as described in claim 1, which is characterized in that the step S5
Middle sequencing result is that bimodal injection batch embryo enters feeding system, and raising is when sexal maturity by screening its next-generation embryo
Detect effective mutation type zebra fish.
5. the method for building up of hepatolenticular degeneration zebra fish model as described in claim 1, which is characterized in that the step S9
The middle micro- sem observation situation method of light field: the juvenile fish that ATP7B is mutated that zebra fish has just hatched in step S5 is taken to be dipped into copper solution
In, light field microscopically observation to zebra fish mutant liver and head nigrescence situation.
6. the method for building up of hepatolenticular degeneration zebra fish model as described in claim 1, which is characterized in that the step S9
The method that middle ICP-MS quantifies copper content: taking 1000 to take, 5 -day-old in step S5 of ATP7B is mutated zebra fish juvenile fish or 5 take
45 -day-old of ATP7B is mutated zebra fish youth fish in step S5, and HClO is added4In 180 DEG C digest 10 hours, after be transferred to 120 DEG C
Shake makes residual acid volatilize, and is diluted with Milli-Q high purity water, measures each tissue copper content with ICP-MS.
7. the method for building up of hepatolenticular degeneration zebra fish model as described in claim 1, it is characterised in that: the step
The analysis of copper sensibility concentration dependant is mutated zebra fish to impregnate ATP7B using various concentration copper-bath in S10, and light field is micro-
The rollover of body that sem observation mutant shows in copper-containing solution, distortion phenotype and for statistical analysis.
8. the method for building up of hepatolenticular degeneration zebra fish model as described in claim 1, which is characterized in that the step
ATP7B is mutated zebra fish juvenile fish and carries out pathological analysis in S10: ATP7B mutation zebra fish juvenile fish being immersed in copper-containing solution, 24
It is sampled after hour, fixed, dehydration, transparent, waxdip, embedding.Slice thickness is 4 μm when slice, HE dyeing, the micro- sem observation of light field
Hepatic pathology situation.
9. the method for building up of the hepatolenticular degeneration zebra fish model as described in claim 5-8, it is characterised in that: described
It is that development is extremely big to 5-7 that ATP7B, which is mutated zebra fish juvenile fish,.
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| CN111514143A (en) * | 2020-05-19 | 2020-08-11 | 杭州师范大学附属医院 | Use of prolyl hydroxylase inhibitors for ameliorating symptoms of hepatolenticular degeneration diseases |
| CN111514143B (en) * | 2020-05-19 | 2021-04-09 | 杭州师范大学附属医院 | Application of prolyl hydroxylase inhibitor in preparation of medicine for improving symptom of hepatolenticular degeneration disease |
| CN113862305A (en) * | 2021-09-17 | 2021-12-31 | 首都医科大学附属北京友谊医院 | Construction method of ATP7B gene knockout mouse model |
| WO2024066542A1 (en) * | 2022-09-27 | 2024-04-04 | 湖南光琇高新生命科技有限公司 | Hepatolenticular degeneration cell, preparation method therefor, and evaluation method therefor |
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